1. Field of the Invention
This invention pertains to the field of modulating gene expression at the transcriptional level. The methods involve tethering a transcriptional coactivator to a DNA binding domain that is specific for a response element, and contacting the coactivator with a transcription factor.
2. Background
Expression of many genes is mediated by signals that increase or decrease transcription of the gene. For example, the presence or absence of a hormone or other compound in or near a cell can switch on or off expression of a particular gene, or family of genes, within the cell. The regulation of gene expression in response to an intracellular or intercellular stimulus is often desirable, such as when such modulation is required for development of the organism or for the ability of the organism to adapt to changes in its environment. However, in some instances gene expression is turned on or off inappropriately. For example, some evidence suggests that estrogen-mediated gene expression is involved in some types of breast and ovarian cancers.
The mechanism by which estrogen and other modulators of transcription exert their effect on gene expression is complex and incompletely understood. In the case of estrogen and other steroid hormones, for example, the hormone binds to a nuclear receptor that is specific for the hormone. The binding of the hormone to the receptor is believed to cause a conformational change that allows the receptor to bind to certain target sites, often referred to as "response elements," that are located upstream of genes that are regulated by the hormone. Additional proteins are involved in regulating transcription mediated by nuclear hormone receptors. The estrogen receptor (ER), for example, binds two classes of coactivator. The first class, referred to as P160s, includes SRC-1a and GRIP-1/TIF-2. These proteins interact with the ligand binding domain (LBD) of nuclear receptors in a manner that is dependent on hormone and the intactness of the LBD trans-activation function, AF2. Onate et al., Science 270: 1354-7 (1995); Voegel et al., EMBO J. 15: 3667-3675 (1996); Hong et al., Proc. Nat'l. Acad. Sci. USA 93: 4948-52 (1996); Halachmi et al., Science 264: 1455-1458 (1994); Cavailles et al., Proc. Natl. Acad. Sci. USA 91: 10009-10013 (1994). The second class consists of CBP and the closely related protein P300, which are required for transcriptional activation by CREB, Jun/Fos and a growing list of transcription factors. Kamei et al., Cell 85: 403-14 (1996); Chakravarti et al., Nature 383: 99-103 (1996); Smith et al., Proc. Nat'l. Acad. Sci. USA 93: 8884-8888 (1996); Hanstein et al., Proc. Nat'l. Acad. Sci. USA 93: 11540-11545 (1996); Janknecht and Hunter, Nature 383: 22-23 (1996). The two types of coactivator are essential for ER-mediated transcriptional activation. Overexpression of members of the p160 family enhance nuclear receptor action, whereas dominant negative SRC-1a and GRIP-1 block nuclear receptor action. Onate et al., supra.; Hong et al., supra. Similarly, overexpression of CBP, or p300, potentiates ER action, whereas micro-injected antibodies against CBP block nuclear receptor action. Kamei et al., supra.; Chakravarti et al., supra. Since the p160s are tightly bound to CBP and p300 in vivo (Kamei et al., supra.; Hanstein et al., supra.), and SRC-1a and CBP bind to both TBP and TFIIB in vitro (Swope et al., J. Biol. Chem. 271: 28138-28145 (1996); Kwok et al., Nature 370: 223-226 (1994), a model has been hypothesized in which the ER works by recruiting the p160-CBP complex to the promoter, and that this complex forms a bridge to the basal transcription machinery (BTM).
These earlier hypotheses do not, however, explain all observations regarding regulation of gene expression by hormones. For example, tamoxifen and related antiestrogens, which are used to treat hormone-dependent breast cancer, bind to the ER and block its activation by estrogen. However, tamoxifen has the undesirable side effect of activating, rather than repressing, estrogen-regulated genes in certain types of cells such as uterine cells. Webb et al., Mol. Endocrinol. 9: 443-456. Promoter regions of genes activated by these antiestrogens were found to have an AP-1 response element, rather than an estrogen response element, but the mechanism by which antiestrogens could exert the paradoxical effect of repressing ER-mediated transcription but activating AP-1-mediated transcription remained unknown. Other estrogen "mimetics" such as certain pesticides and other chemicals may provide an inappropriate signal for certain genes to be expressed or repressed in an undesirable manner, resulting in "feminization" of males. These estrogen mimetics can act like estrogens even though the mimetics appear to be chemically unrelated to estrogen. Other chemicals may inappropriately stimulate or repress the effects of other classes of signaling molecules, such as neurotransmitters and growth factors.
Although methods for modulating gene expression would be highly valuable in treating many conditions, the development of such methods has been hampered by a lack of understanding of the mechanisms by which moieties such as nuclear hormone receptors exert their effects on gene expression. Therefore, a need exists for methods for modulating gene expression, and for identifying compounds that are capable of stimulating or repressing signal-mediated changes in gene expression. The present invention fulfills these and other needs.